Integrated structural biology of the native malarial translation machinery and its inhibition by an antimalarial drug

Abstract

Our understanding of cellular events is hampered by the gap between the resolution at which we can observe events inside cells and our ability to replicate physiological conditions in test tubes. Here, we show in Plasmodium falciparum, a non-model organism of high medical importance, that this gap can be bridged by using an integrated structural biology approach to visualize events inside the cell at molecular resolution. We determined eight high-resolution structures of the native malarial ribosome in actively translating states inside P. falciparum-infected human erythrocytes using in situ cryo-electron tomography. Following perturbation with a Plasmodium-specific translation inhibitor, we then observed a decrease in elongation factor-bound ribosomal states and an apparent upregulation of ribosome biogenesis in inhibitor-treated parasites. Our work elucidates new molecular details of the malarial translation elongation cycle and demonstrates direct multiscale visualization of drug-induced phenotypic changes in the structure and localization of individual molecules within the native cellular context.

Fig. 1. Single-particle Pf80S ribosomes.

a–c, Single-particle cryo-EM reconstructions, at 2.4–2.8-Å resolution, of the Pf80S ribosome in the POST state with P-site and E-site occupied, with the L1 stalk indicated (a), non-rotated PRE state with P-site, E-site and A-site occupied (b) and rotated PRE state with P-site and E-site occupied (c). The map is colored as follows: 60S large subunit, light gray; 40S small subunit, dark gray; A-site, P-site and E-site tRNAs, green, cyan and yellow, respectively. LSU, large subunit; SSU, small subunit.

Fig. 2. Visualizing molecular details of P. falciparum asexual intraerythrocytic life-cycle stages using in situ cryo-ET.

a,b, In situ cryo-ET workflow from sample preparation to data analysis. Parasite-infected red blood cells (iRBCs) were applied to a grid support and plunge-frozen in liquid ethane (L_E) (a), followed by cryo-correlative light and EM (CLEM) imaging showing distribution of fluorescently labeled parasites (b). c, Top-down view of a frozen grid in a cryo-FIB-SEM. Blue arrowheads, schizont iRBCs. d,e, FIB images of the sample thinned to ~3 µm (d) and the final thinned cross-section (lamella) at ~150 nm (e). f, Montaged cryo-TEM image of the entire lamella. White arrowheads, parasites; black arrowheads, contamination; white star, milling artifacts. g, Averaged central slice of a tomogram reconstructed from tilt series collected on lamella. h, Ultrastructural segmentations and STA Pf80S ribosomes mapped back into the tomographic volume and overlaid onto the corresponding central slice from g. i, STA consensus reconstruction of the Pf80S ribosome at an overall resolution of 4.1 Å. j, Pf80S consensus map, shown in cross-section and colored according to local resolution, as calculated in RELION3.1. k, Ribbon models of 28S rRNA and eL43 ribosomal protein segments, shown with corresponding STA density (surface representation).

Fig. 3. Ensemble of in situ Pf80S ribosomes comprising the malarial translation elongation cycle.

a–i, Native structures of the P. falciparum translational machinery, determined by in situ cryo-ET, elucidate the molecular ensemble of Pf80S ribosomes in malaria parasites. Directions of the small subunit rotation, backroll and head swivel are indicated on a cartoon of an 80S ribosome relative to the large subunit. Translation intermediate states (a–h) and unloaded ribosomal states (i) distinguished by position of principal ligands in these high-resolution STA reconstructions, enabling reconstitution of the P. falciparum translation elongation cycle. The refined map for each state is reconstructed from all particles corresponding to a particular class across datasets. Maps are shown in cross-section for clarity. eEF1α, dark red; A-tRNA, green; L1 stalk movement, pink; A/P-tRNA, teal; P-tRNA, cyan; eEF2, salmon; E-tRNA, orange.

Fig. 4. Molecular effects of CBQ treatment revealed by thermal PISA, integrated cell montages and quantitative proteomics.

a, Graph of percentages of EF-bound states in CBQ-treated and DMSO control parasites, across four time points and four technical replicates. Percentages are based on total identified ribosomes after particle curation (n = 230,471). b, Volcano plot of differential abundance of soluble proteins in parasites treated with CBQ, relative to the DMSO vehicle control. Each protein is represented as a function of the log₂ fold change (x axis) and −log₁₀ P value (y axis) based on moderated t-test analysis carried out for each gradient independently. Hit selection cutoffs of P < 0.01 and a log₂ fold change of >0.2 are represented by dashed lines. Drug-stabilized proteins, red; destabilized proteins, blue; proteins exhibiting no significant (NS) abundance change, gray. c, Relative soluble protein abundance of eEF2 and eEF1α identified in a. Protein abundance under CBQ treatment is plotted relative to the DMSO vehicle in each respective thermal gradient (n = 4 biological replicates). Significance changes (moderated t-test) are represented by asterisks (**P < 0.01 and ***P < 0.001). eEF2, P = 0.00037; eEF1α, P = 0.000064. Error bars represent the s.d. d, Integrated cell montage of a CBQ-treated parasite frozen, milled and imaged at 18 h after treatment. Segmented features are color-coded as indicated by corresponding colored labels. Mapped back ribosomes are colored as follows: orange, monomers; pale yellow, dimers and polysomes; red, membrane-bound ribosomes. e, Volcano plot of differentially expressed proteins in CBQ-treated parasites comparing 36 h to 18 h after treatment (52 versus 38 hpi). The mean average of each protein from three replicates (n = 3) is shown. The y axes show the log₁₀ P value and x axes show the log₂ fold change using a nested study design. Blue and red circles indicate proteins with significant change. Gray circles indicate proteins with no significant (P > 0.05) change in expression. Hit selection cutoffs of P < 0.05 and log₂ fold change > 0.1 are represented by dashed lines. f, REVIGO-generated GO enrichment analysis of enriched proteins in CBQ-treated parasites at 36 h compared to 18 h. Bars are colored by REVIGO-designated cluster. A one-way ANOVA and pairwise comparison were used to compare each condition. The y axes represent the log₁₀ number of annotated GO terms in P. falciparum. Each bar represents a subcluster.

Extended Data Fig. 1. Single particle data processing scheme and refined structures of the Pf80S ribosome.

a, Five states were separated as displayed in the pictured classification scheme, using the following masks: small-subunit (yellow) for separating rotated from non-rotated states, P-site (pink) for non-rotated particles, P-site (blue) for rotated particles and A-site (green) for non-rotated particles. b-f, Final post-processed maps of the five Pf80S ribosome states, shown in cross section. Maps are colored as follows: 60S large subunit in light grey, 40S small subunit in dark grey, mRNA in dark orange, and the A-, P-, and E-site tRNAs in green, cyan, and yellow, respectively. g-k, Corresponding cross-sectioned view of each Pf80S map is shown colored according to local resolution, as calculated in RELION.

Extended Data Fig. 2. 48 h time course following treatment with CBQ at 20hpi.

Hema3-stained blood smears of highly synchronized early trophozoite-stage P. falciparum parasites, taken at 0, 1, and 3 h after addition of DMSO, CBQ, or CHX at 20hpi, and every 4^th hour after, out to 48 h. Three biological replicates were performed, with n = 25-81 for each time point in each replicate. One representative replicate is shown. Time of drug addition at 20hpi and time points corresponding to those used in our in situ cryoET studies are indicated with a green and black arrowheads, respectively. Box with a bold pink outline indicates a pyknotic cell.

Extended Data Fig. 3. The P. falciparum asexual intraerythrocytic life cycle.

a, Schematic of the P. falciparum life cycle, with focus on the intraerythrocytic asexual replication stages in human red blood cells (RBCs). b, Representative images, from 12 independent experiments, of Hema3-stained, parasite-infected RBCs (iRBCs) at the different intraerythrocytic stages from invasion to replication drawn from (n = 25-81). Averaged central slice of a tomogram reconstructed from tilt series collected on lamella.

Extended Data Fig. 4. Focused classification of the subtomogram averaged native Pf80S from consensus reconstruction.

a, Translation intermediates were separated following the displayed scheme using the following masks: elongation factor (EF) (green), PE-tRNA (magenta), EF-A-P-PE-E binding sites (cyan), P-tRNA (purple). For all panels the number of particles, percentage of total particles and resolution are indicated below each map. b, Fourier shell correlation (FSC) curve for ribosomal states identified through classification to determine global resolution using the ‘Gold-standard’ 0.143 cut off (dotted line). Identified ribosomal states are shown in descending order from highest to lowest resolution in the left side of the graph. c, Averaged central slice of a tomogram reconstructed from tilt series collected on lamella.

Extended Data Fig. 5. Details of subtomogram averaged maps and perturbations of the translational landscape under CBQ-treatment.

a, Examples of nascent chain density visible in the exit tunnel of POST, decoding-1 and decoding-2 and unloaded states. b, Bar graphs showing the translational landscape comprised of relative distribution of identified translation intermediates and ribosomal states in CBQ-treated and DMSO-control parasites. Percentages are based on total amount of identified ribosomes after particle curation. c-d, Bar graph showing the percentage of elongation factor-bound states across 4 time points in CBQ-treated and DMSO- control parasites, across four technical replicates. Percentages are based on total amount of identified ribosomes after particle curation (n = 230,471). Error bars represent SD from the mean. e, Relative soluble protein abundance of four CBQ-stabilized tRNA synthetases identified in the DIA Thermal PISA experiment: Arginine tRNA Ligase (ArgRS), Alanine tRNA Ligase (AlaRS), Leucine tRNA Ligase (LeuRS), Tryptophan tRNA Ligase (TrpRS). Protein abundance levels under CBQ treatment are plotted relative to the DMSO vehicle in each respective thermal gradient (n = 4 biological replicates). Significance changes (moderated t-test) are represented by asterisks (p value < 0.01 ‘**’; < 0.001 ‘***’). Error bars represent ± SD.

Extended Data Fig. 6. Details of integrated cell montages of CBQ- and DMSO-treated P. falciparum parasites and quantitative proteomic analysis.

a, Integrated cell montage of a CBQ-treated parasite frozen, milled and imaged at 3 h post-treatment. Averaged central slices from tomograms collected on the cell are shown mapped onto a tracing of the cell, with ultrastructural segmentations and mapped back ribosome reconstructions overlaid on top. Segmented features are color coded as indicated by corresponding colored labels. Mapped back ribosomes are colored as follows: orange=monomers, pale-yellow=dimers and polysomes, red=membrane bound ribosomes. b-c, Integrated cell montage of a DMSO-vehicle parasite frozen, milled and imaged at 3 h (b) and 18 h (c) post-treatment. Segmentation is colored as in (a). c, Detailed view of the averaged central slices overlaid with segmentations containing the DV of the cell shown in (a). Segmented features are color coded as in (a). PVM, parasitophorous vacuolar membrane. PPM, parasite plasma membrane. EMS, endomembrane system. DV, digestive vacuole. Hz, hemozoin. ER, endoplasmic reticulum. d, Total count of number of nuclei containing Pf60S at 3 and 18 h under DMSO-vehicle and CBQ-treatment. e, Hema3-stained smears of parasites at 3, 18 and 36 h after addition of CBQ or DMSO- vehicle (23, 38, and 56 hpi). Parasites treated with DMSO-vehicle have egressed and reinvaded by the 36 hr (56 hpi) time point from three biological and technical replicates. f, Volcano plot showing differentially expressed proteins under CBQ-pressure comparing proteins present at 36 h to 3 h post treatment (56 to 23 hpi). The mean average of each protein from three replicates (n = 3) is shown. Y-axes show log10 p- value and x-axes show log₂ fold change using nested study design. Dark coral and blue circles indicate proteins with significant enrichment and depletion, respectively. Grey circles indicate proteins with no significant (p > 0.05) change in expression. Hit selection cut-offs of p < 0.05 and log2 Fold-change of >0.1 are represented by dashed lines. One way ANOVA and pairwise comparison was used to compare each condition.